An optimally functioning immune system requires a balance between protective responses against environmental pathogens and tolerogenic responses to innocuous environmental antigens. The breakdown of tolerance to harmless environmental antigens results in antigens becoming allergens that induce allergic inflammation and disease. Regulatory T cells (Treg) are central to the development and maintenance of tolerance to allergens; however, the mechanisms by which Treg fail to support tolerance in patients with allergic disease are not fully defined. In this application, our preliminary data strongly support that a host's inability to signal through the prostaglandin (PG)I2 receptor impedes immune tolerance and that treatment with PGI2 analogs promotes tolerance. Specifically, we found that defective PGI2 signaling in thymic Treg (tTreg) resulted in decreased Foxp3 expression, reduced suppressive function, inhibited IL-10 production, and downregulated inducible Treg (iTreg) differentiation. We also showed that Treg from mice deficient in the PGI2 receptor IP (IP KO) mice did not suppress type 2 cytokine production in an in vivo adoptive transfer model of allergic inflammation compared to Treg from WT mice. Therefore, our overarching hypothesis is that PGI2 promotes the immunosuppressive functions of Treg. To test this hypothesis, we have two specific aims. Specific Aim 1 is to determine the ability of endogenous PGI2 signaling to promote Treg function. In this aim we hypothesize that: a) antigen-specific Treg that have deficient PGI2 signaling have inhibited suppression of the cardinal features of asthma compared to antigen-specific WT Treg, b) PGI2 signaling promotes Treg stability in the setting of allergen challenge, and c) PGI2 signaling restrains ST2 expression on Treg, and that a deficiency of PGI2 signaling corrupts Treg function promoting the development of pathogenic IL-13 expressing ST2+ Treg. Specific Aim 2 is to determine the ability of exogenous PGI2 signaling to promote Treg function. In this aim we hypothesize that: a) PGI2 analog treatment of Treg ex vivo enhances the ability of iTreg to restrain allergic inflammation, b) exogenous administration of a PGI2 analog increases Treg function to suppress allergic inflammation, and c) PGI2 analogs enhance IL-10 production and suppressive function by human iTreg in response to activation with IL-2 and TGF-?. These studies are paradigm shifting because there are currently no known pharmacologic agents which promote Treg function and our preliminary data strongly supports that PGI2 may be the first described. The proposed experiments using human cells and mice will advance the field in that they will further define the mechanisms by which Treg function is regulated. The current availability of PGI2 for human treatment highlights the clinical significance of our studies as this therapy could be immediately repurposed for the treatment of allergic respiratory diseases such as asthma.